CTL epitopes from EBV

ABSTRACT

The present invention provides cytotoxic Epstein-Barr virus (EBV) T-cell epitopes derived from EBV structural antigens. Preferred epitopes include YLLEMLWRL (SEQ ID NO: 1), YFLEILWGL (SEQ ID NO: 32), YLLEILWRL (SEQ ID NO: 33), YLQQNWWTL (SEQ ID NO: 6), LLLALLFWL (SEQ ID NO: 2), LLVDLLWLL (SEQ ID NO: 3), LLLIALWNL (SEQ ID NO: 4), WLLLFLAIL (SEQ ID NO: 5), TLLVDLLWL (SEQ ID NO: 7), LLWLLLFLA (SEQ ID NO: 8), ILLIIALYL (SEQ ID NO: 9), VLFIFGCLL (SEQ ID NO: 10), RLGATIWQL (SEQ ID NO: 11), ILYFIAFAL (SEQ ID NO: 15), SLVIVTTFV (SEQ ID NO: 17), LMIIPLINV (SEQ ID NO: 20), TLFIGSHVV (SEQ ID NO: 24), LIPETVPYI (SEQ ID NO: 26), VLQWASLAV (SEQ ID NO: 27) and QLTPHTKAV (SEQ ID NO: 29). The present invention also provides methods of treating or preventing EBV infection in subjects which involve administration of EBV cytotoxic T-cell epitopes.

FIELD OF THE INVENTION

[0001] The present invention relates to methods of treating orpreventing EBV infections. The present invention also relates tocytotoxic T-cell (CTL) epitopes within Epstein-Barr virus (EBV)structural and latent antigens, and to subunit vaccines and nucleic acidvaccines which include these epitopes.

BACKGROUND OF THE INVENTION

[0002] It is now well established that long-term protection frompersistent viral infection requires the development of virus-specificmemory T cells which recognize viral antigens in association with eitherclass I or class II MHC molecules. Since immunization with whole viralproteins is unable to elicit an efficient CTL response, interest hasbeen directed towards designing vaccines based on defined epitopesequences. This is particularly the case with oncogenic viruses, sinceindividual viral genes introduced in recombinant vectors have thepotential to initiate tumorgenic processes. Two broad approaches arecurrently being considered to design an effective vaccine forcontrolling Epstein-Barr virus (EBV) associated diseases (for review seeref. (7)). These include directing immune responses to either EBVstructural antigens or latent antigens.

[0003] In the last few years, most of the vaccine development effortshave concentrated on the use of a subunit preparation of gp350(recombinant and affinity purified) and have been directed towardsblocking virus attachment to the target cell in the oropharynx (31). Thegeneral approach has been to immunize cotton-top marmosets with gp350and determine their ability to restrict the outgrowth of EBV-positivelymphomas in these animals. Indeed, highly purified gp350, whenadministered subcutaneously in conjunction with adjuvants (muramyldipeptide or ISCOMS), induced high levels of serum neutralizingantibodies and inhibited tumor formation in cotton-top tamarins (32). Anumber of recombinant vectors including, vaccinia-gp350 and adenovirus5-gp350 have also been successfully used in these animals to block tumoroutgrowth (33). The precise mechanism by which gp350 affords protectionfrom lymphomas in cotton-top tamarins remains unclear. The fact thatdevelopment of neutralizing antibody titres in vaccinated animals doesnot always correlate with protection indicates that gp350-specific Tcell-mediated immune responses may also have an effector role (34).Furthermore, Yao and colleagues (35) showed that very low levels ofneutralizing anti-gp350 antibodies are present in the saliva of healthyEBV-immune donors, which suggests that such antibodies are unlikely tobe the basis of long-term inmmunity in healthy seropositive individuals.It has been postulated that gp350 specific T cell-mediated immuneresponses may have an effector role in protection. There has been noidentification to date, however, of CTL epitopes within the EBVstructural antigens.

[0004] Post-transplant lymphoproliferative disease (PTLD) that arises inorgan transplant patients is an increasingly important clinical problem.Histological analysis of PTLD shows a quite complex clonal diversityranging from polymorphic B lymphocyte hyperplasia to malignantmonoclonal lymphoma. This range of pathology encompasses the collectiveterm PTLD while the lymphomas are frequently referred to asimmunoblastic lymphomas(IL). This condition is clearly associated withthe proliferation of Epstein-Barr virus (EBV) infected B cells which arecarried for life in all previously infected individuals (about 80% ofadults and 20% of children 7 years) (45, 46, 47, 49). These EBV-infectedB cells are normally restricted in their growth in vitro and in vivo byvirus-specific cytotoxic T cells (CTLs) which recognise epitopesincluded within the EBV latent proteins (see below) (48).Immunosuppression inhibits these specific CTL and results in anexpansion of the pool of EBV-infected B cells and the emergence of theclinical problems associated with PTLD. It is known that the individualsat greatest risk of PTLD are EBV seronegative recipients who receive atransplant from a seropositive donor (Crawford and Thomas, 1993).Immunisation of EBV seronegative graft recipients prior to engraftmentwill greatly reduce the risk of PTLD.

[0005] The role of the immunue system in the rejection ofvirus-associated cancers has also been the subject of intense studyrecently. The hypothesis under investigation is that many neoplasmsexpress viral antigens that should potentially enable them to berecognized and destroyed by the immune system, including both T helpercells and cytotoxic T lymphocytes (CTL). There is now compellingevidence that most of the Epstein-Barr virus (EBV)-associatedmalignancies escape this potent virus-specific CTL response byrestricting viral gene expression (7.20,21). For malignancies such asnasopharyngeal carcinoma (NPC) and Hodgkin's disease (HD), EBV nuclearantigen 1 (EBNA1) and latent membrane protein 1 (LMP1) are the onlyantigens consistently expressed and are therefore the potential targetantigens for any future vaccine designed to control these tumors (3,28).Since it is well established that immunization with whole viral proteinsdoes not elicit an efficient CTL response, interest has been directedtowards developing peptide vaccines based on defined epitope sequences.

SUMMARY OF THE INVENTION

[0006] Results obtained by the present inventors indicate that CTLepitopes within EBV structural and latent proteins may be effective inproviding antiviral immunity against EBV infection. In particular, thepresent inventors have analysed the latent antigen LMP1 sequence, usingpeptide stablization assays, and found that this antigen includespotential CTL epitopes. Following in vitro activation with thesepeptides, both polyclonal and clonal CTLs from HLA A2-positive donorsshowed strong reactivity against target cells expressing the LMP1antigen. Moreover, lymphoblastoid cell lines (LCL), expressing differentHLA A2 supertypes were efficiently recognized by these CTLs, a resultthat has important implications for the design of an anti-viral vaccineaimed at protecting different ethnic populations.

[0007] The present inventors have also found that CTLs from acuteinfectious mononucleosis (IM) patients display strong reactivity againstthe EBV structural antigens gp85 and gp350. In addition, specific CTLepitopes within EBV structural antigens gp85 and gp350 have beenidentified for the first time. Importantly, prior immunisation of HLAA2/K^(b) transgenic mice with these gp350 and gp85 CTL epitopes induceda strong epitope-specific CTL response and afforded protection againstgp85- or gp350-expressing vaccinia virus challenge. These resultsprovide evidence, for the first time, of the existence of CTL epitopesin EBV structural proteins and show that they may be used forestablishing strong anti-viral immunity against EBV infection.

[0008] Accordingly, in a first aspect the present invention provides acytotoxic Epstein-Barr virus (EBV) T-cell epitope, the epitope beingderived from an EBV structural antigen.

[0009] In a preferred embodiment of the first aspect of the presentinvention, the EBV structural antigen is gp85 or gp350.

[0010] In a second aspect the present invention provides a cytotoxicEpstein-Barr virus T-cell epitope, the epitope being selected from thegroup consisting of YLLEMLWRL (SEQ ID NO: 1), YFLEILWGL (SEQ ID NO: 32),YLLEILWRL (SEQ ID NO: 33), YLQQNWWTL (SEQ ID NO: 6), LLLALLFWL (SEQ IDNO: 2), LLVDLLWLL (SEQ ID NO: 3), LLLIALWNL (SEQ ID NO: 4), WLLLFLAIL(SEQ ID NO: 5), TLLVDLLWL (SEQ ID NO: 7), LLWLLLFLA (SEQ ID NO: 8),ILLIIALYL (SEQ ID NO: 9), VLFIFGCLL (SEQ ID NO: 10), RLGATIWQL (SEQ IDNO: 11), ILYFIAFAL (SEQ ID NO: 15), SLVIVTTFV (SEQ ID NO: 17), LMIIPLINV(SEQ ID NO: 20), TLFIGSHVV (SEQ ID NO: 24), LIPETVPYI (SEQ ID NO: 26),VLQWASLAV (SEQ ID NO: 27) and QLTPHTKAV (SEQ ID NO: 29).

[0011] In a third aspect the present invention provides a subunitvaccine including a cytotoxic Epstein-Barr virus (EBV) T-cell epitopeaccording to the first aspect of the present invention.

[0012] In a preferred embodiment, the subunit vaccine includes at leastone T-cell epitope selected from the group consisting of YLLEMLWRL (SEQID NO: 1), YFLEILWGL (SEQ ID NO: 32), YLLEILWRL (SEQ ID NO: 33),YLQQNWWTL (SEQ ID NO: 6), LLLALLFWL (SEQ ID NO: 2), LLVDLLWLL (SEQ IDNO: 3), LLLIALWNL (SEQ ID NO: 4), WLLLFLAIL (SEQ ID NO: 5), TLLVDLLWL(SEQ ID NO: 7), LLWLLLFLA (SEQ ID NO: 8), ILLIIALYL (SEQ ID NO: 9),VLFIFGCLL (SEQ ID NO: 10), RLGATIWQL (SEQ ID NO: 11), ILYFIAFAL (SEQ IDNO: 15), SLVIVTTFV (SEQ ID NO: 17), LMIIPLINV (SEQ ID NO: 20), TLFIGSHVV(SEQ ID NO: 24), LIPETVPYI (SEQ ID NO: 26). VLQWASLAV (SEQ ID NO: 27)and QLTPHTKAV (SEQ ID NO: 29).

[0013] In a preferred aspect of the present invention the epitope isselected from the group consisting of YLLEMLWRL (SEQ ID NO: 1),YLQQNWWTL (SEQ ID NO: 6), YFLEILWGL (SEQ ID NO: 32), YLLEILWRL (SEQ IDNO: 33), SLVIVTTFV (SEQ ID NO: 17), LMIIPLINV (SEQ ID NO: 20), TLFIGSHVV(SEQ ID NO: 24), and VLQWASLAV (SEQ ID NO: 27).

[0014] In a further preferred embodiment, the subunit vaccine includesone or more additional cytotoxic EBV T-cell epitopes. The additionalcytotoxic EBV T-cell epitope(s) may be selected from those described inWO 97/45444, the entire contents of which are incorporated herein byreference.

[0015] In a further preferred form of the present invention the vaccineincludes a water-in-oil formulation. It is further preferred that thevaccine includes at least one antigen to which the individual will mountan anamnestic response in addition to the at least one cytotoxic T-cellepitope.

[0016] The at least one antigen is preferably selected from the groupconsisting of tetanus toxoid. diphtheria toxoid, Bordetella pertussisantigens, poliovirus antigens, purified protein derivative (PPD), gp350protein (Thorley-Lawson, D. A. and Poodry, G. A. (1982). Identificationand isolation of the main component (gp350-gp220) of Epstein-Barr virusresponsible for generating neutralizing antibodies in vivo. J. Virol.43, 730-736), helper epitopes and combinations thereof and is preferablytetanus toxoid.

[0017] It is preferred that the water-in-oil formulation is MontanideISA 720. Additional information regarding this formulation can be foundin WO 95/24926, the disclosure of which is incorporated herein by crossreference.

[0018] The subunit vaccine may also be formulated using ISCOMs. Furtherinformation regarding ISCOMs can be found in Australian Patent Nos.558258, 590904, 632067, 589915, the disclosures of which are includedherein by cross reference.

[0019] In a fourth aspect the present invention provides an isolatednucleic acid sequence encoding a cytotoxic Epstein-Barr virus (EBV)T-cell epitope according to the first aspect of the present invention.

[0020] In a preferred embodiment, the isolated nucleic acid sequenceencodes at least one of the cytotoxic T-cell epitopes selected from thegroup consisting of YLLEMLWRL (SEQ ID NO: 1), YFLEILWGL (SEQ ID NO: 32),YLLEILWRL (SEQ ID NO: 33), YLQQNWWTL (SEQ ID NO: 6), LLLALLFWL (SEQ IDNO: 2), LLVDLLWLL (SEQ ID NO: 3), LLLIALWNL (SEQ ID NO: 4), WLLLFLAIL(SEQ ID NO: 5), TLLVDLLWL (SEQ ID NO: 7), LLWLLLFLA (SEQ ID NO: 8),ILLIIALYL (SEQ ID NO: 9), VLFIFGCLL (SEQ ID NO: 10), RLGATIWQL (SEQ IDNO: 11), ILYFIAFAL (SEQ ID NO: 15), SLVIVTTFV (SEQ ID NO: 17), LMIIPLINV(SEQ ID NO: 20), TLFIGSHVV (SEQ ID NO: 24), LIPETVPYI (SEQ ID NO: 26),VLQWASLAV (SEQ ID NO: 27) and QLTPHTKAV (SEQ ID NO: 29).

[0021] As will be appreciated by those skilled in the field the nucleicacid sequence can be delivered as naked nucleic acid or using a suitableviral or bacterial vectors. Suitable bacterial vectors include thebacteria Salmonella spp. Suitable viral vectors include, for example,retroviral vectors, adenoviral vectors and vaccinia vectors. An exampleof a suitable vaccinia vector is a modified Vaccinia Ankara vector.

[0022] Vectors suitable for delivery of nucleic acid sequences havepreviously been described. For example, alphavirus vectors have becomewidely used in basic research to study thestructure and function ofproteins and for protein production purposes. Development of a varietyof vectors has made it possible to deliver foreignsequences as naked RNAor DNA, or as suicide virus particles produced using helper vectorstrategies. Preliminary reports also suggest that these vectors may beuseful for in vivo applications where transient, high-level proteinexpression is desired, such as recombinant vaccines. The initial studieshave already shown that alphavirus vaccines can induce strong humoraland cellular immune responses with good immunological memory andprotective effects. See, for example, Tubulekas I., Berglund P., FleetonM., and Liljestrom P. (1997) Alphavirus expression vectors and their useas recombinant vaccines: a minireview, Gene 190(1):191-195.

[0023] Recombinant pox viruses have been generated for vaccinationagainst heterologous pathogens. Amongst these, the following are notableexamples. (i) The engineering of the Copenhagen strain of vaccinia virusto express the rabies virus glycoprotein. When applied in baits, thisrecombinant has been shown to vaccinate the red fox in Europe andraccoons in the United States, stemming the spread of rabies virusinfection in the wild. (ii) A fowlpox-based recombinant expressing theNewcastle disease virus fusion and hemagglutinin glycoproteins has beenshown to protect commercial broiler chickens for their lifetime when thevaccine was administered at 1 day of age, even in the presence ofmaternal immunity against either the Newcastle disease virus or the poxvector. (iii) Recombinants of canarypox virus, which is restricted forreplication to avian species, have provided protection against rabiesvirus challenge in cats and dogs, against canine distemper virus, felineleukemia virus, and equine influenza virus disease. In humans, canarypoxvirus-based recombinants expressing antigens from rabies virus. Japaneseencephalitis virus, and HIV have been shown to be safe and immunogenic.(iv) A highly attenuated vaccinia derivative, NYVAC. has been engineeredto express antigens from both animal and human pathogens. Safety andimmunogenicity of NYVAC-based recombinants expressing the rabies virusglycoprotein, a polyprotein from Japanese encephalitis virus, and sevenantigens from Plasmodium falciparum have been demonstrated to be safeand immunogenic in early human vaccine studies. See, for example,Paoletti E. (1996) Applications of pox virus vectors to vaccination: anupdate, Proc Natl Acad Sci USA, 93(21):11349-11353.

[0024] Progress towards effective vaccines to control internalparasites, especially those affecting mucosal compartments, has beeninhibited by the combined problems of the antigenic complexity ofparasites and the lack of understanding of the host response. However,the accumulation of information regarding regulation of mucosal immunityhas enabled a reappraisal of vaccination options to provide appropriatemucosal effector responses. The pivotal role of T cell influences, andin particular the contribution of cytokine signals, has been clearlyestablished from in vitro studies, but data emerging from ourlaboratories provide evidence for these effects in vivo. We havedemonstrated the role of T cells in determining the outcome of anintestinal response and propose a role for local Th2 cytokine productionin this regard. To support this proposition, the distribution ofcytokine mRNA has been determined by in situ hybridisation techniques innormal and parasitised animals. Further, we have shown that in theabsence of Th2 cytokines (using gene knockout animals) mucosal responsesare grossly deficient; we have also shown that this defect can beovercome by vector-directed gene therapy. These studies have indicatedthat new mucosal immunisation opportunities exist by combiningtraditional immunisation approaches with strategies to upregulate localcytokine production. However, the success of these new strategies willdepend on selection of highly immunogenic subunit antigens, coupled withtechniques for cytokine manipulation and delivery with appropriateadjuvant/vehicle formulations. This paper reviews delivery technologiesavailable to chaperone labile antigenic and genetic material toappropriate sites for mucosal stimulation after systemic or oraladministration. See, for example, Sutter G. et al (1994) A recombinantvector derived from the host range-restricted and highly attentuated MVAstrain of vaccinia virus stimulates protective immunity in mice toinfluenza virus. Vaccine 12:1032-1040; and Husband A. J., Bao S.,McClure S. J., Emery D. L., Ramsay A. J. (1996) Antigen deliverystrategies for mucosal vaccines, Int J Parasitol 8-9:825-834.

[0025] Attenuated Salmonella typhi vaccine strain CVD 908, which harborsdeletion mutations in aroC and aroD, has been shown to be well-toleratedand highly immunogenic, eliciting impressive serum antibody, mucosal IgAand cell-mediated immune responses. A further derivative prepared byintroducing a deletion in htrA (which encodes a heat-shock protein thatalso has activity as a serine protease in CVD 908 resulted in CVD908-htrA. In phase 1 clinical trials, CVD 908-htrA appears veryattractive as a live oral vaccine candidate. Both CVD 908 and CVD908-htrA are useful as live vector vaccines to deliver foreign antigensto the immune system. Conditions that enhance the expression andimmunogenicity of foreign antigens carried by CVD 908 and CVD 908-htrAare being investigated. For a review of Salmonella vectors, see LevineM. M., Galen J., Barry E., Noriega F., Chatfield S., Sztein M., DouganG. And Tacket C (1996) Attenuated Salmonella as live oral vaccinesagainst typhoid fever and as live vectors, J Biotechnol 44(1-3):193-196.

[0026] The isolated nucleic acid sequences may be in the form of nucleicacid vaccines. Further information regarding nucleic acid vaccines canbe found in WO 96/03144 and in Suhrbier A (1997), Multi-epitope DNAvaccines, Immunol Cell Biol 75(4):402-408 the disclosures of which areincorporated herein by cross reference.

[0027] In a fifth aspect the present invention provides an isolatedpolypeptide, the polypeptide including at least one epitope according tothe first or second aspects of the present invention.

[0028] The vaccines of the present invention may be usedprophylactically or therapeutically.

[0029] The CTL epitopes of the present invention may be synthesisedusing techniques well known to those skilled in this field. For example,the CTL epitopes may be synthesised using solution synthesis or solidphase synthesis as described, for example, in Chapter 9 entitled“Peptide Synthesis” by Atherton and Sheppard which is included in apublication entitled “Synthetic Vaccines” edited by Nicholson andpublished by Blackwell Scientific Publications. Preferably a solid phasesupport is utilised which may be polystyrene gel beads wherein thepolystyrene may be cross-linked with a small proportion ofdivinylbenzene (e.g. 1%) which is further swollen by lipophilic solventssuch as dichloromethane or more polar solvents such as dimethylformamide(DMF). The polystyrene may be functionalised with chloromethyl oranionomethyl groups. Alternatively, cross-linked and functionalisedpolydimethyl-acrylamide gel is used which may be highly solvated andswollen by DMF and other dipolar aprolic solvents. Other supports can beutilised based on polyethylene glycol which is usually grafted orotherwise attached to the surface of inert polystyrene beads. In apreferred form, use may be made of commercial solid supports or resinswhich are selected from PAL-PEG, PAK-PEG, KA, KR or TGR.

[0030] In solid state synthesis, use is made of reversible blockinggroups which have the dual function of masking unwanted reactivity inthe a-amino, carboxy or side chain functional groups and of destroyingthe dipolar character of amino acids and peptides which render theminactive. Such functional groups can be selected from t-butyl esters ofthe structure RCO—OCMe₃—CO—NHR which are known as t-butoxy carboxyl orROC derivatives. Use may also be made of the corresponding benzyl estershaving the structure RCO—OCH₂—C₆H₅ and urethanes having the structureC₆H₅CH₂O CO—NHR which are known as the benzyloxycarbonyl orZ-derivatives. Use may also be made of derivatives of fluorenyl methanoland especially the fluorenyl-methoxy carbonyl or Fmoc group. Each ofthese types of protecting group is capable of independent cleavage inthe presence of one other so that frequent use is made, for example, ofBOC-benzyl and Fmoc-tertiary butyl protection strategies.

[0031] Reference also should be made to a condensing agent to link theamino and carboxy groups of protected amino acids or peptides. This maybe done by activating the carboxy group so that it reacts spontaneouslywith a free primary or secondary amine. Activated esters such as thosederived from p-nitrophenol and pentafluorophenyl may be used for thispurpose. Their reactivity may be increased by addition of catalysts suchas 1-hydroxybenzotriazole. Esters of triazine DHBT (as discussed on page215-216 of the abovementioned Nicholson reference) also may be used.Other acylating species are formed in situ by treatment of thecarboxylic acid (i.e. the Nα-protected amino acid or peptide) with acondensing reagent and are reacted immediately with the amino component(the carboxy or C-protected amino acid or peptide).Dicyclohexylcarbodiimide, the BOP reagent (referred to on page 216 ofthe Nicholson reference), O'Benzotriazole-N,N, N′N′-tetra methyl-uroniumhexaflurophosphate (HBTU) and its analogous tetrafluroborate arefrequently used condensing agents.

[0032] The attachment of the first amino acid to the solid phase supportmay be carried out using BOC-amino acids in any suitable manner. In onemethod BOC amino acids are attached to chloromethyl resin by warming thetriethyl ammonium salts with the resin. Fmoc-amino acids may be coupledto the p-alkoxybenzyl alcohol resin in similar manner. Alternatively,use may be made of various linkage agents or “handles” to join the firstamino acid to the resin. In this regard, p-hydroxymethyl phenylacticacid linked to aminomethyl polystyrene may be used for this purpose.

[0033] As will be readily appreciated by those skilled in the art theLMP1, gp85 and gp350 epitopes and vaccines of the present invention canbe used to treat and to protect against EBV. Further, given the possiblegreater involvement of EBV infection in immunocompromised individuals,the present invention may have particular application in the treatmentand protection of individuals having decreased immune function, egtransplant patients. Importantly, the present inventors have found thatEBV transformed lymphoblastoid cell lines expressing different HLA A2supertypes are efficiently recognised by LMP1-specific CTL clones. Thishighlights the potential for the design of an antiviral vaccine aimed attreating and protecting different ethnic populations.

[0034] Accordingly, in a sixth aspect the present invention provides amethod of preparing a composition for use in inducing CTLs in a subject,the method including admixing at least one epitope according to thefirst or second aspects of the present invention with at least onepharmaceutical acceptable carrier, diluent or excipient.

[0035] In a seventh aspect the present invention provides a method ofreducing the risk of EBV infection in a subject which method includesadministering to the subject an effective amount of:

[0036] (1) at least one CTL epitope according to the first or secondaspects of the present invention;

[0037] (2) a subunit vaccine according to the third aspect of thepresent invention;

[0038] (3) a nucleic acid sequence according to the fourth aspect of thepresent invention;

[0039] (4) a vector according to the fourth aspect of the presentinvention; or

[0040] (5) a polypeptide according to the fifth aspect of the presentinvention.

[0041] In an eighth aspect the present invention provides a method oftreating or preventing nasopharyngeal carcinoma or Hodgkin's disease ina subject which method includes administering to the subject aneffective amount of at least one CTL epitope derived from an EBVstructural or latent antigen.

[0042] By “effective amount” we mean a quantity of the epitope which issufficient to induce or amplify a CTL response against an EBV antigen.

[0043] In a preferred embodiment of the eighth aspect of the presentinvention, the EBV structural antigen is gp85 or gp350 and the latentantigen is LMP1 or LMP2. In a further preferred embodiment the CTLepitope is an epitope as defined in the second aspect of the presentinvention.

[0044] The present inventors have also made the surprising finding thatNPC cells which are recognised by CTL clones are subject to CTL lysis.This finding has important implications for the design of vaccines tocontrol NPC tumours in vivo.

[0045] In an ninth aspect the present invention provides a method oftreating or preventing growth of NPC or HD cells in a subject in needthereof which method includes administering to the subject at least oneCTL epitope derived from an EBV structural or latent antigen.

[0046] In a preferred embodiment of the ninth aspect of the presentinvention, the EBV CTL epitopes are derived from the gp85 or gp350antigens. In a further preferred embodiment, the EBV CTL epitopes arederived from the LMP1 or LMP2 antigens. Preferably, the CTL epitopes arederived from the LMP1 antigen.

[0047] In a tenth aspect the present invention provides a method oftreating or preventing the growth of NPC or HD cells in a first subjectwhich method includes transferring to the subject EBV-specific CTLswhich recognise NPC or HD cells.

[0048] In a preferred embodiment the EBV-specific CTLs are obtained fromNPC patients by in vitro stimulation of CTLs by exposure to EBV CTLepitopes. Alternatively, the EBV-specific CTLs may be obtained from asecond subject, wherein the second subject is infected with EBV but doesnot have NPC.

[0049] In a further preferred embodiment of the tenth aspect of thepresent invention, the EBV-specific CTLs are LMP1 and/or LMP2-specificCTLs.

[0050] In an eleventh aspect the present invention provides a method ofreducing the risk of infectious mononucleosis or post transplantationlymphoproliferative disease in a subject which method includesadministering to the subject an effective amount of:

[0051] (1) at least one CTL epitope according to the first or secondaspects of the present invention;

[0052] (2) a subunit vaccine according to the third aspect of thepresent invention;

[0053] (3) a nucleic acid sequence according to the fourth aspect of thepresent invention;

[0054] (4) a vector according to the fourth aspect of the presentinvention; or

[0055] (5) a polypeptide according to the fifth aspect of the presentinvention.

[0056] The terms “comprise”, “comprises” and “comprising” as usedthroughout the specification are intended to refer to the inclusion of astated component or feature or group of components or features with orwithout the inclusion of a further component or feature or group ofcomponents or features.

[0057] In order that the nature of the present invention may be moreclearly understood forms thereof will now be described with reference tothe following examples and figures.

BRIEF DESCRIPTION OF THE FIGURES

[0058]FIG. 1: MHC stablization analysis on T2 cells using potential HLAA2 binding peptides within LMP1. T2 cells were initially incubated with200 μl of each of the peptides (200 μg/ml) for 14-16 h at 26° C.followed by incubation at 37° C. for 2-3 h. HLA A2 expression on thesecells was analysed by FACS using the BB7.2 antibody. The dotted lineindicates the background mean fluorescence intensity for HLA A2 on T2cells without any peptide. The LMP1 peptides showing significantstablization of HLA A2 molecules on T2 cells are indicated by arrows.

[0059]FIG. 2: Recognition of LMP1 peptides by polyclonal CTLs from anHLA A2-positive donor SB. PBMC from donor SB were co-cultivated forseven days with irradiated T2 cells sensitized with synthetic peptides(indicated on the Y-axis). On day 10, these cells were used aspolyclonal effectors in a standard ⁵¹Cr-release assay againstpeptide-sensitized (1 μg/ml) autologous PHA blasts. An effector:targetratio of 20:1 was used in the assay. Data from one representativeexperiment out of three is shown. Results are expressed as percentspecific lysis.

[0060]FIG. 3: Specific lysis by an EBV-specific CTL clone (SB7) fromdonor SB of autologous LCLs and autologous CD40 B cells infected withVacc.EBNA1, 2, 3, 4, 5, 6, LMP1, LMP2A and Vacc.TK−(panel A). Targetcells were infected for 12-14 h (M.O.I=10:1) with vaccinia constructsand processed for the standard ⁵¹Cr-release assay. Vacc.TK− was used asa control recombinant vaccinia. To further confirm the LMP1 specificityof the SB7 clone, LMP1-and HLA A2-positive BL cell lines (BJAB.MTLM6 orMutu cl.59) and LMP1-negative, HLA A2-positive BL cell lines (BJAB.gpt1and Mutu cl.216) were used as targets in a standard ⁵¹Cr-release assay(panel B). An effector:target ratio of 4:1 was used in both assays.Results are expressed as percent specific lysis.

[0061]FIG. 4: CTLp frequencies for the LMP1 epitopes YLQQNWWTL (SEQ IDNO: 6) (A) and YLLEMLWRL (SEQ ID NO:1) (B) in HLA A2-positive donor areshown. Using limiting dilution analysis, the frequencies of CTLp forpeptides YLQQNWWTL (SEQ ID NO: 6) (A) and YLLEMLWRL (SEQ ID NO: 1) (B)were estimated in peripheral blood lymphocytes from donor SB. PBMC fromthis donor was stimulated with peptides sensitized PBMC as described inthe “Materials and Methods” section. Reciprocal values of responderfrequencies (f¹) are indicated. The shaded areas indicate 95% confidencelimits.

[0062]FIG. 5: CTL recognition of LCLs expressing different supertypes ofHLA A2 by the SB7 CTL clone. All LCLs used in this assay weretransformed with the B95.8 EBV isolate. The HLA A2 subtyping for eachLCL was assigned by the 12th Histocompatability workshop. Aneffector:target ratio of 4:1 was used in both the assay. Results areexpressed as percent specific lysis. Data from one representativeexperiment out of four is shown.

[0063]FIG. 6: Effect of variant LMP1 peptides on the HLA A2 binding(panel A) and CTL recognition (panel B). For HLA A2 binding analysis onT2 cells were initially incubated with 200 μl of each of the peptides(200 μg/ml) for 14-16 h at 26° C. followed by incubation at 37° C. for2-3 h. HLA A2 expression on these cells was analysed by FACS using BB7.2antibody (panel A). The dotted line indicates the background meanfluorescence intensity for HLA A2 on T2 cells without any peptide. ForCTL recognition of the variant and prototypic HLA A2-restricted LMP1epitope. PHA blasts from donor SB were sensitized with serial dilutionsof each of the peptides and then exposed to the SB7 CTL clone (panel B)at an effector:target ratio of 4:1. Results are expressed as percentspecific lysis. Data from one representative experiment out of three isshown. Amino acid changes in the variant peptides are indicated by boldletters.

[0064]FIG. 7: CTL recognition of NPC cells and LCLs by EBV-specific HLAA11-restricted CTL clones CM9 (a & c) and CM29 (b & d). Vacc.EBNA4,Vacc.TK infected or peptide sensitized C15NPC cells (a & b) were exposedto CTL clones at different effector to target ratios and the level ofCTL lysis was compared to a type 2 LGLs (CM/Ag876 LCL). As a positivecontrol target cells were either presensitized with IVTDFSVIK peptide (a& c), or AVFDRKSDAK peptide (b & d). The results were expressed as percent specific lysis.

[0065]FIG. 8: MHC stabilization analysis on T2 cells using potential HLAA2 binding peptides within gp85 (panel A) and gp350 (panel B). T2 cellswere initially incubated with 200 Tl of each of the peptides (200 Tg/ml)for 14-16 h at 26° C. followed by incubation at 37° C. for 2-3 h. HLA A2expression on these cells was analysed by FACS using the BB7.2 antibody.The dotted line indicates the background mean fluorescence intensity forHLA A2 on T2 cells without any peptide. The gp85 and gp350 peptidesshowing significant stabilization of HLA A2 molecules on T2 cells areindicated by arrows.

[0066]FIG. 9: gp85 and gp350-specific ex vivo cytotoxic T cell activityin peripheral blood lymphocytes from IM donors. Panel A, B & C shows exvivo CTL lysis of peptide-sensitized (1 μg/ml) PHA blasts at twodifferent effector:target (E/T) ratios. Data from IM patient SB, LP andMG are presented in panel A, B and C respectively. Panel D shows ex vivoCTL lysis by peripheral blood lymphocytes from patient LP of targetcells infected with recombinant vaccinia encoding either gp85(Vacc.gp85) or gp350 (Vacc.350) by. Vacc.TK− and Vacc.EBNA2 were used ascontrol in the assay.

[0067]FIG. 10: Recognition of gp85 and gp350 peptides by polyclonal CTLsfrom an HLA A2-positive IM recovered (36 months post IM) individual.PBMC were co-cultivated for seven days with irradiated T2 cellssensitized with synthetic peptides (indicated on the Y-axis). On day 18,these cells were used as polyclonal effectors in a standard 51Cr-releaseassay against peptide-sensitized (1 Tg/ml) autologous PHA blasts. Aneffector:target ratio of 20:1 was used in the assay. Individual peptidestimulated CTL effectors used in this experiments are indicated in thefigure. Data from one representative experiment out of three is shown.Results are expressed as percent specific lysis.

[0068]FIG. 11: Immunization of HLA A2/K^(b) mice with gp85 and gp350 CTLepitopes induces strong CTL response. Animals were twice immunized (at a14 day interval) subcutaneously with individual CTL epitope with TetanusToxoid as a source of help. Four weeks following peptide immunization,animals were assessed for gp350- and gp85-specific CTL response. PanelA, B & C shows CTL activity in splenocytes from two mice immunized withVLQWASLAV (SEQ ID NO: 27) (gp350), TLFIGSHVV (SEQ ID NO: 24) (gp85) andSLVIVTTFV (SEQ ID NO: 17) (gp85) respectively. CTL lysis of target cellssensitized with peptide epitopes are shown as filled symbols, whilelysis of unsensitized target cells is shown as empty symbols. Panel Dshows CTL activity in pooled inguinal lymphnodes from mice immunisedwith peptide VLQWASLAV (SEQ ID NO: 27) (▪, □), SLVIVTTFV (SEQ ID NO: 17)(▴, Δ) and TLFIGSHVV (SEQ ID NO: 24) (, ◯). CTL activity was tested onday 6 using a standard 51Cr-release assay.

[0069]FIG. 12: Prior immunisation of HLA A2/Kb mice with gp85 or gp350CTL epitopes affords protection against recombinant vaccinia viruschallenge. Groups of female A2/Kb transgenic mice, either unimmunized orimmunized with CTL epitopes, were challenged with Vacc.gp85 andVacc.gp350 intraperitonealy. After four days of challenge, these animalswere sacrificed and vaccinia titre measured in both ovaries by plaqueassay on confluent CV1 cells. X-axis shows peptides used forimmunization, while Y-axis shows Mean+/−SE vaccinia virus titre in naiveand peptide immunized mice. Recombinant vaccinia virus used forchallenge in these animals is shown in each panel of the figure.

DETAILED DESCRIPTION OF THE INVENTION EXAMPLE 1 Materials and Methods

[0070] Establishment and Maintenance of Cell Lines

[0071] LCLs were established from sero-positive donors by exogenousvirus transformation of peripheral B cells using the B95.8 (Type 1) orAg876 (Type 2) virus isolates. In addition, LCLs transformed with theB95.8 isolate and expressing different HLA A2 supertypes were also usedin this study (12th Histocompatability workshop cell panel; EACC). Thepeptide transporter (TAP)-negative B×T hybrid cell line 174×CEM.T2(referred to as T2) (22) were used for peptide stablization assays. Allcell lines were routinely maintained in RPMI 1640 containing 2 mMglutamine, 100 IU/ml penicillin and 100 ug/ml streptomycin plus 10%foetal calf serum (FCS) (growth medium). Long-term cultures ofEBV-negative normal B cell blasts were established as previouslydescribed using the CD40 system (referred to as CD40 B cells) (12).

[0072] The Burkitt's lymphoma (BL) cell lines, BJAB.gpt1, BJAB.MTLM6,MUTU cl.59 and MUTU cl.216 were used in this study. These were derivedfrom patients with non-endemic or endemic BL. BJAB.MTLM6 and MUTU cl.59have previously been shown to express LMP1, while BJAB.gpt1 and MUTUcl.216 are negative for LMP1 (5,27). These BL cell lines were routinelymaintained in growth medium.

[0073] To generate phytohaemagglutinin (PHA) blasts, peripheral bloodmononuclear (PBMC) cells were stimulated with PHA (Commonwealth SerumLaboratories, Melbourne) and after 3 days, growth medium containing MLA144 supernatant and rIL-2 was added (2). PHA blasts were propagated withbi-weekly replacement of IL-2 and MLA supernatant (no further PHA added)for up to 6 weeks.

[0074] Virus Isolates

[0075] To isolate resident EBV, spontaneous LCLs were established from apanel of 12 unrelated healthy EBV-seropositive donors by spontaneousoutgrowth from PBMC cultured in the presence of 0.1 μg/ml cyclosporin A(19). In addition, virus isolates from eight different NPC samples (fromSoutheast Asia) were directly sequenced from biopsy material. Theseisolates were classified as type 1 EBV based on the DNA sequencedivergence within the BAM H1 WYH and E regions of the genome (23,24).

[0076] PCR and DNA Sequencing of CTL Epitopes

[0077] Specific oligonucleotide primers flanking the DNA region encodingthe LMP1 epitope were selected for PCR amplification. The resulting PCRproducts were purified using QIAquick spin columns (Qiagen Inc.Chatsworth, Calif.) and sequenced in both directions using a PRISM readyreaction dyedeoxy terminator cycle sequencing kit (Applied BiosystemsInc., Foster City, Calif.) following the manufacturer's protocol.

[0078] Synthesis of Peptides

[0079] Peptides synthesized by the Merrifield solid phase method (16)were purchased from Chiron Mimotopes (Melbourne, Australia). dissolvedin dimethyl sulphoxide and diluted in serum-free RPMI 1640 medium foruse in standard CTL assays.

[0080] MHC Stabilisation Assays

[0081] To identify the potential HLA A2 binding peptides within LMP1, acomputer based program was employed as described elsewhere (http://bimas.dcrt.nih.gov/molbio/hla_bind/index.html) (18). These predictedpeptides were then used in a standard MHC stablization assay using T2cells as described earlier (1). Briefly, T2 cells (2×10⁵) were incubatedwith 200 μl of each of the peptides (200 μg/ml) at 26° C. for 14-16 h,followed by incubation at 37° C. for 2-3 h. After the incubations, HLAA2 expression was measured by FACS using a monoclonal HLA A2-specificantibody (MA2.1; ATCC).

[0082] Generation of Polyclonal and Clonal LMP1-Specific CTLs

[0083] To generate polyclonal CTLs, PBMC from HLA A2-positive donorswere co-cultivated for seven days with the irradiated (8,000 rads) T2cells sensitized with synthetic peptides. On day 7, these lymphocyteswere restimulated with peptide-sensitized T2 cells. After 10 days ofculture in growth medium, the cells were used as polyclonal effectors ina standard ⁵¹Cr-release assay against peptide-sensitized autologous PHAblasts.

[0084] To generate LMP1-specific CTL clones, PBMC (10⁶/ml) werecultivated with peptide sensitized autologous lymphocytes (responder tostimulator ratio of 4:1) in 2 ml culture wells (Linbro) for 3 days ingrowth medium. CTL clones, generated by seeding in 0.35% agarose, wereestablished and maintained in growth medium containing highly purifiedrecombinant human IL-2 from E. coli (16), restimulating twice weeklywith autologous LCLs. These CTL clones were screened on a panel ofrecombinant vaccinia-infected autologous CD40 B cells to confirm theantigen specificity (see below).

[0085] Vaccinia Virus Recombinants

[0086] Recombinant vaccinia constructs encoding EBV latent antigens anda vaccinia virus construct made by insertion of the pSC11 vector aloneand negative for thymidine kinase (Vacc.TK−) have been previouslydescribed (6.11). CD40 B cells were infected with recombinant vacciniavirus at a multiplicity of infection (MOI) of 10:1 for 1 h at 37° C. asdescribed earlier (6.11). After overnight infection, cells were washedwith growth medium and processed for CTL assays or for immunoblotting toassess the expression of recombinant EBV antigens (12).

[0087] Cytotoxicity Assay

[0088] Target cells were either infected with recombinant vacciniaviruses or pre-sensitized with synthetic peptide epitopes (wild-type orvariant) and then incubated with ⁵¹Cr for 90 min. Following incubation,these cells were washed in growth medium and used as targets in astandard 5 h ⁵¹Cr-release assay (16). In some experiments, monoclonalantibodies (MoAb) specific for the non-polymorphic determinants on MHCclass I (W6/32) or class II (L243) antigens were added to define the MHCrestriction of the CTL clones.

[0089] Limiting Dilution Analysis (LDA)

[0090] PBMC from HLA A2-positive donors were distributed in gradednumbers (two fold dilutions) from 6.25×10³ to 5×10⁴ cells per well inround-bottomed microtiter plates. Approximately 5×10⁴ K-irradiated(2,000 rads) peptide sensitized (1 μg/ml) auologous PBMC were added togive a total volume of 100 μl. Twenty-four replicates were used at eachconcentration in each experiment. Cultures were fed on days 4 and 7 with50 μl of medium supplemented with 20U of rIL-2 and 30% (vol/vol)supernatant from MLA-144 cultures. On day 10, each CTL microculture wassplit into two replicates and used as effectors in a standard 5 h⁵¹Cr-release assay against autologous PHA blasts precoated with an LMP1peptide or left uncoated. Wells were scored as positive when the percentspecific chromium release for peptide-sensitized target cells exceededthe mean release from untreated control wells by 3 SDs. LDA wasperformed by the method of maximum likelihood estimation (4). Data fromall experiments were compatible with the hypothesis of single-hitkinetics (P>0.4) and precursor estimates are given with 95% confidencelimits.

Results

[0091] Identification of HLA A2 Binding Peptides within LMP1

[0092] To identify potential HLA A2-restricted epitopes within LMP1. theamino acid sequence was analyzed by a computer program designed topredict HLA-binding peptides, based on an estimation of the half-timedisassociation of the HLA-peptide complex(http://bimas.dcrt.nih.gov/molbio/hla_bind/index.html) (18). A total of11 peptides with an estimated half-time disassociation score of >400were selected (Table 1). These peptides were then tested for HLA A2binding efficiency using HLA A2-positive T2 cells. Representative datafrom a series of experiments is presented in FIG. 1. This analysisshowed that six peptides significantly increased the expression of HLAA2 on T2 cells suggesting that these peptides bind to this allele.

[0093] Generation of Polyclonal CTLs Specific LMP1 Peptides

[0094] The data presented above strongly suggested that LMP1 includessequences which can bind HLA A2 molecules and are therefore potentialtargets for virus-specific CTLs. To verify this hypothesis, PBMC fromtwo HLA A2-positive EBV immune donors (SB and AS) were stimulated withT2 cells sensitized with each of the HLA A2-binding peptides from LMP1.On day 10, these effector cells were tested against peptide-sensitizedautologous PHA blasts. Represtative data from polyclonal CTLs from donorSB are presented in FIG. 2. Two peptides, YLQQNWWTL (SEQ ID NO: 6) andYLLEMLWRL (SEQ ID NO: 1), showed significant activation of polyclonalCTLs; however, the YLLEMLWRL-stimulated CTLs consistently showedsignificantly stronger CTL activity when compared to theYLQQNWWTL-stimulated cells. Similar data were also obtained for anotherHLA A2-positive donor (AS) (data not shown). TABLE 1 Identification ofPotential HLA A2 Binding Peptides within LMP1* Score (Estimate half-timedisassociation from HLA Ranking Residue Peptide Sequence A2 allele) 1125-133 YLLEMLWRL (SEQ ID NO:1) 25714.76 2 32-40 LLLALLFWL (SEQ ID NO:2)9858.69 3 167-175 LLVDLLWLL (SEQ ID NO:3) 2568.45 4  92-100 LLLIALWNL(SEQ ID NO:4) 2317.87 5 173-181 WLLLFLAIL (SEQ ID NO:5) 1302.88 6159-167 YLQQNWWTL (SEQ ID NO:6) 1252.90 7 166-174 TLLVDLLWL (SEQ IDNO:7) 999.86 8 171-179 LLWLLLFLA (SEQ ID NO:8) 935.11 9 152-160ILLIIALYL (SEQ ID NO:9) 739.03 10 110-118 VLFIFGCLL (SEQ ID NO:10)510.60 11 132-140 RLGATIWQL (SEQ ID NO:11) 441.60

[0095] Charaterization of LMP1 CTL Epitope

[0096] To further characterize the CTL epitopes identified by thepolyclonal CTLs, virus-specific CTL clones were generated using initialstimulation with peptide sensitized autologous PBMC followed bycontinous restimulation with irradiated autologous LCLs. Proliferatingclones were screened for peptide recognition using the rapid visualassay for CTL specificity (2) and one clone, SB7, clearly recognized theYLLEMLWRL (SEQ ID NO: 1) peptide. No CTL clones specific for YLQQNWWTL(SEQ ID NO: 6) were isolated using this procedure. To further confirmthe antigen specificity of the SB7 clone, autologous CD40-stimulated Bcells were infected with recombinant vaccinia viruses encodingindividual EBV antigens and then exposed to these CTLs. The datapresented in FIG. 3A clearly demonstrate that only target cells infectedwith the LMP1-expressing vaccinia construct were recognized. Inaddition, only LMP1- and HLA A2-positive BL cell lines (BJAB.MTLM6 orMutu cl.59) were efficiently lysed by this clone, while BL cellsnegative for this antigen (BJAB.gpt1 and Mutu cl.216) were notrecognized (FIG. 3B). These results confirm that YLLEMLWRL (SEQ IDNO: 1) is an LMP1 CTL epitope that is endogenously processed byvirus-infected cells.

[0097] In the next set of experiments we analyzed the frequency of CTLprecursors (CTLp) for this epitope in PBMC from HLA A2-positive healthyEBV immune donors by limiting dilution analysis. YLLEMLWRL-specific CTLpwere reactivated in vitro by stimulation of PBMCs from donors SB and ASwith autologous peptide-sensitized PBMCs. Autologous PHA blastsprecoated with peptide YLLEMLWRL (SEQ ID NO: 1) or untreated were usedas target cells in a chromium release assay. The representative data inFIG. 4 show that a very low frequency of memory CTL specific for theYLLEMLWRL (SEQ ID NO: 1) epitope were detected in the HLA A2-positivedonors AS (1:223,535+/−107,437) and SB (1:252,650+/−122,875).

[0098] To determine whether EBV-transformed LCLs expressing differentHLA A2 supertypes could be recognized by clone SB7, a panel of LCLsexpressing ten different supertypes of the HLA A2 allele (HLA A*0201-HLAA*0210) were screened in a standard CTL assay. The data presented inFIG. 5 clearly demonstrate that LCLs expressing all the major HLA A2supertypes except HLA A*0205 were efficiently recognized by the CTLclone SB7. This lysis was significantly inhibited by the HLA classI-specific antibody W6/32. Surprisingly, HLA A2-positive andTAP-negative T2 cells were also recognized by this clone suggesting thatthe YLLEMLWRL (SEQ ID NO: 1) epitope is endogenously processed through aTAP-independent pathway (FIG. 5).

[0099] Sequence Analysis of the HLA A2-Restricted LMP1 Epitope in VirusIsolates from NPC Patients and Healthy Donors

[0100] Efficient presentation of the LMP1 epitope by HLA A*0201, HLAA*0203 and HLA A*0207, which are common supertypes in the SoutheastAsian ethnic population, raised the possibility that this epitope mightbe important as a potential target epitope for LMP1-expressing NPC.Sequence analysis across this CTL epitope region in virus isolates fromeight NPC samples was carried out using LMP1-specific primers.Spontaneous LCLs from healthy EBV immune donors were used as controls inthis analysis. Interestingly all EBV isolates from the NPC samplesdisplayed identical substitutions within this epitope (Table 2).

[0101] In contrast, of the 12 virus isolates from healthy EBV immunedonors, four encoded a sequence identical to that of the B95.8 isolate,while six displayed a different pattern of alterations within thisepitope that differed from those found in the NPC samples. In someisolates leucine at position 2, methionine at position 5 and arginine atposition 8 were substituted with phenylalanine, isoleucine and glycine,respectively (Table 2), while in other isolates only the methionine atposition 5 was substituted with isoleucine. Importantly, incubation ofT2 cells with these variant peptides (YFLEILWGL (SEQ ID NO: 32) andYLLEILWRL (SEQ ID NO: 33)) significantly increased MHC expression onthese cells (FIG. 6A), indicating efficient binding to HLA A2. theYLLEILWRL (SEQ ID NO: 33) peptide was efficiently recognized by the SB7clone, while no CTL activity was seen in the presence of the YFLEILWGL(SEQ ID NO: 32) peptide (FIG. 6B). The latter result does not rule outthe possibility that YFLEILWGL (SEQ ID NO: 32) is an epitope individualsinfected with an EBV strain encoding this sequence. The loss ofrecognition by the clone SB7 is likely to be due an inappropriate T cellreceptor interaction with the MHC-peptide complex rather than loss ofMHC binding. Thus it is possible that T cells expressing a different Tcell receptors are capable of efficiently recognising this HLA bindingpeptide (8). Interestingly, virus isolates from the other two healthyEBV immune donors TABLE 2 Sequence of HLA A2-restricted LMP1 epilope(YLLEMLWRL) in EBV isolates from NPC and healthy seropositiveindividuals. Number HLA A2^(b) of Virus Origin Epilope Sequence^(a)Binding Isolates B95.8 ⁹⁶⁶⁷⁶TAC TTA TTG GAG ATG CTC TGG CGA CTT⁹⁶⁷⁰²++++ Y L L E M L W R NPC Southeast --- --C --- --- --T --- --- --- ---Asia {open oversize bracket} -  F  -  -  I  -  -  -  -  − 8 HealthyCaucasian --- --- --- --- --- --- --- --- --- donors {open oversizebracket} -  -  -  -  -  -  -  -  -  ++++ 4 Caucasian --- --- --- --- --T--- --- --- --- ++++ 2 {open oversize bracket} -  -  -  -  I  -  -  - -  Caucasian --- --C --- --- --T --- --- G-G --- ++++ 4 {open oversizebracket} -  F  -  -  I  -  -  G  -  Caucasian --- --C --- --- --T ------ --- --- − 1 {open oversize bracket} -  F  -  -  I  -  -  -  - Southeast --- --C --- --- --T --- --- --- --- − 1 Asia {open oversizebracket} -  F  -  -  I  -  -  -  - 

[0102] (including one from Southeast Asia) displayed changes within theepitope sequence which were identical to those seen in the NPC samples(Table 2).

[0103] Analysis of Antigen Processing Function of NPC Cells

[0104] To determine whether NPC cells can present endogenously expressedantigens to virus-specific CTLs, tumour cells were either infected withrecombinant vaccinia encoding EBNA4 (Vacc.EBNA4) or presensitized withsynthetic peptide epitopes. FIG. 7 illustrates the results from anexperiment in which recombinant vaccinia-infected NPC cells (C15) werecompared with HLA-matched type 2 LCLs infected with Vacc.EBNA4.Following exposure to EBNA4-specific CTLs, Vacc.EBNA4-infected orpeptide sensitized NPC cells were efficiently recognised by both CM19and CM29 CTL clones. The level of CTL lysis was comparable to that seenfor LCLs. These results clearly demonstrate that NPC cells are able totransport sufficient levels of peptides into the ER by TAP-dependentmechanism and can efficiently transport MHC-peptide complexes from theER to the surface of the cell.

[0105] Normal antigen processing function in NPC cells has significantimplications for vaccines designed to control these tumours in vivo.Earlier studies have demonstrated that the latent gene expression in NPCis often limited to EBNA1 and the transmembrane proteins, LMP1 and LMP2(29).

[0106] Since EBNA1 is not recognized by EBV-specific CTLs, there is anincreasing emphasis on designing strategies to control NPC aroundepitopes known to be included within LMP1 (6,17). In view of the datapresented in this study, it is reasonable to assume that LMP epitopeswill be processed efficiently by NPC cells. An effective approach tocontrol NPC cells in vivo may be to amplify LMP-specific CTL responsesin these patients. This might be achieved by two different procedures.Firstly, NPC patients might be immunised with synthetic peptides whichinclude CTL epitopes from LMP1 and/or LMP2. Alternatively. LMP1 andLMP2-specific CTLs from HLA matched healthy virus carriers may beadoptively transferred into NPC patients in a manner analogous to thatused to successfully treat EBV-associated polyclonal lymphomas in bonemarrow transplant recipients (30).

Discussion

[0107] Earlier work from various laboratories have shown that the viralphenotype of EBV-associated malignancies is likely to be a veryimportant factor in reducing tumor susceptibility to virus-specific CTLsurveillance, since viral antigen expression in these malignant cells invivo is restricted to either EBNA1, or EBNA1 and LMP1 (9,20,21). Sinceit is now firmly established that EBNA1 does not include classI-restricted CTL epitopes (6,17), considerable interest has beendirected towards identifying potential epitopes within LMP1. The presentstudy was precisely designed to address this issue. One of the limitingfactors in identifying epitopes within LMP1 has been the fact that theCTL response to this antigen often constitutes as a minor component ofthe total virus-specific response (6,17). To overcome this problem weemployed a modified protocol to identify potential HLA A2-restrictedepitopes within LMP1. An important step in this process was the use of acomputer based program developed by Parker and colleagues (18) designedto predict the potential HLA binding peptides within various proteinsfrom human pathogens. Analysis of the LMP1 sequence from the B95.8 EBVisolate revealed a number of potential HLA A2 binding peptides and alarge proportion of these were then functionally shown to stabilize HLAA2 molecules on T2 cells. Stimulation of PBMCs with these peptidesresulted in the activation of a strong polyclonal CTL response specificfor the peptide YLLEMLWRL (SEQ ID NO: 1), while a weaker response wasseen for another peptide YLQQNWWTL (SEQ ID NO: 6). The YLLEMLWRL (SEQ IDNO: 1) sequence was confirmed as an LMP1 epitope by isolating a CTLclone (SB7) specific for this peptide. The LMP1 specificity of the SB7CTL clone was further confirmed by the recombinant vaccinia experimentsand efficient lvsis of LMP1 expressing HLA A2-positive BL cells.

[0108] The CTL response characterized in the present report is ofinterest not only because it is directed against a viral antigenconstitutively expressed in many EBV-associated malignancies (HD andNPC) but also because the HLA A2 allele is common in virtually all humanpopulations (13). More importantly, EBV transformed LCLs expressing allthe major HLA A2 supertypes were efficiently recognized by theLMP1-specific CTL clone, a result that has important implications foranti-viral vaccine design aimed at protect different ethnic populations.It is important to mention here that the CTL, response to the LMP1epitope in healthy seropositive individuals constitutes a minorcomponent of the virus-specific CTL response and very low levels of CTLprecursors are seen for this epitope. It may nevertheless be possible toamplify this component by vaccination with the relevant peptide or withadoptive transfer of in vitro activated LMP1-specific CTLs. Suchapproaches may be of use in the control of HD and NPC.

[0109] Efficient presentation of the YLLEMLWRL (SEQ ID NO: 1) peptide byHLA A*0201, HLA A*0203 and HLA A*0207-positive LCLs, which are commonsupertypes in the southeast Asian population, raises the possibilitythat this epitope might be exploited as a potential target epitope forLMP1-expressing NPC.

EXAMPLE 2 Materials and Methods

[0110] Infectious Mononcleosis (IM) Patients

[0111] IM patients, identified on clinical grounds and by heterophileantibody positivity, were bled during the first 5-10 days of illnessand, in two cases, on a second occasion 24-36 months after theresolution of symptoms. These patients were HLA typed for the HLA A2allele by serotyping in microcytotoxicity and by genotyping. Threepatients (SB, LP and MG) were identified as HLA A2-positive patients andthis was subsequently confirmed by FACS analysis using an HLAA2-specific monoclonal antibody (ATCC).

[0112] Establishment and Maintenance of Cell Lines:

[0113] EBV-transformed lymphoblastoid cell lines (LCLs) were establishedfrom a panel of IM and healthy EBV-seropositive donors by exogenousvirus transformation of peripheral B cells using type 1 (B95.8) or type2 (Ag876) EBV isolates (16), and were routinely maintained in RPMI 1640containing 2 mM glutamine, 100 IU/ml penicillin and 100 ug/mlstreptomycin plus 10% foetal calf serum (FCS) (growth medium). Inaddition, the peptide transporter (TAP)-negative B×T hybrid cell line174×CEM.T2 (referred to as T2) (22) were used for peptide stablizationassays.

[0114] To generate phytohaemagglutinin (PHA) blasts peripheral bloodmononuclear cells (PBMC) were stimulated with PHA (Commonwealth SerumLaboratories, Melbourne) and after 3 days, growth medium containing MLA144 supernatant and rIL-2 was added (36). PHA blasts were propagatedwith bi-weekly replacement of IL-2 and MLA supernatant (no further PHAadded) for up to 6 weeks.

[0115] Establishment and Preparation of CTL Effectors

[0116] Acute IM PBMC effectors for use in ex vivo cytotoxicity assayswere resuspended in growth medium supplemented with recombinant IL2 andused directly in a cytotoxicity assay (see below). To generatepolyclonal CTLs, PBMCs from HLA A2-positive donors were co-cultivatedfor seven days with irradiated (8,000 rads) T2 cells presensitized withsynthetic peptides (37). On days 7 and 14, these cultures wererestimulated with peptide-sensitized T2 cells. After 18 days of culturein growth medium, the cells were used as polyclonal effectors in astandard 51Cr-release assay against peptide-sensitized autologous PHAblasts.

[0117] Synthesis of Peptides

[0118] Peptides, synthesized by the Merrifield solid phase method, werepurchased from Chiron Mimotopes (Melbourne, Australia), dissolved indimethyl sulphoxide, and diluted in serum-free RPMI 1640 medium for usein standard CTL assays.

[0119] MHC Stabilisation Assays

[0120] HLA A2 binding peptides within the gp85 and gp350 antigens wereidentified using a protocol as described in Example 1. These predictedpeptides were then used in a standard MHC stablization assay using T2cells. Briefly, T2 cells (2×10⁵) were incubated with 200 μl of each ofthe peptides (200 μg/ml) at 26° C. for 14-16 h, followed by incubationat 37° C. for 2-3 h. After the incubations. HLA A2 expression wasmeasured by FACS using a monoclonal HLA A2-specific antibody (MA2.1:ATCC).

[0121] Vaccinia Virus Recombinants

[0122] Recombinant vaccinia constructs encoding the EBV structuralantigens gp350 (Vacc.gp350) and gp85 (Vacc.gp85), and a vaccinia virusconstruct made by insertion of the pSC11 vector alone and negative forthymidine kinase (Vacc.TK−) have been previously described (38). Targetcells were infected with recombinant vaccinia virus at a mutiplicity ofinfection (MOI) of 10:1 for 1 h at 37° C., as described earlier (6,12).After overnight infection, cells were washed with growth medium andprocessed for CTL assays or for immunoblotting to assess the expressionof recombinant EBV antigens (11).

[0123] Cytotoxicity Assay

[0124] Target cells were either infected with recombinant vacciniaviruses or pre-sensitized with synthetic peptide epitopes and thenincubated with 51Cr for 90 min. Following incubation, these cells werewashed in growth medium and used as targets in a standard 5 h51Cr-release assay (16).

[0125] Immunisation of HLA A2/Kb Transgenic Mice with gp350 and gp85 CTLEpitopes

[0126] HLA A2/K b transgenic mice used in this study have been describedelsewhere (39). These mice express a chimeric class I molecule composedof the alpha 1 & 2 domains of the human A*0201 allele and the alpha 3domains of the mouse H-2Kb class I molecules. Peptide immunizations werecarried out as described by Vitello and colleagues (40). Briefy, theseanimals were twice immunized (at a 14 day interval) subcutaneously with50 ug/mouse of CTL epitopes emulsified in IFA together with 5 ug ofTetanus Toxoid as a source of help. Four weeks following peptideimmunization, animals were assessed for gp350- and gp85-specific CTLresponse. For assessing these CTL responses, splenocytes (3×10⁶cells/ml) were cocultured with syngeneic, irradiated (2000 rad)peptide-coated LPS blasts (3×10⁵ cells/ml) and 3 ug/ml humanB2-microglobulin. CTL activity was tested on day 6 using a standard51Cr-release assay.

[0127] Vaccinia Protection Assay

[0128] For protection experiments, groups of 8 weeks old female A2/Kbtransgenic mice were immunized with CTL epitopes as described above. Onday 28, mice were challenged with Vacc.gp85 and Vacc.gp350intraperitonealy (1×10⁷ pfu in 100 ul PBS). After four days ofchallenge, these animals were sacrificed and vaccinia titres measured inboth ovaries by plaque assay on confluent CV1 cells.

Results

[0129] Identification of HLA A2 Binding Peptides within gp85 and gp350

[0130] To identify potential HLA A2-restricted epitopes within gp8S andgp350, the amino acid sequence was analyzed by a computer programdesigned to predict HLA-binding peptides, based on an estimation of thehalf-time disassociation of the HLA-peptide complex(http://bimas.dcrt.nih.gov/molbio/hla_bind/index.html) (18). A total of20 peptides (13 from gp85 and 7 from gp350) with an estimated half-timedisassociation score of >100 for gp85 and >50 for gp350 were selected(Table 3). These peptides were then tested for HLA A2 binding efficiencyusing HLA A2-positive T2 cells. Representative data from a series ofexperiments is presented in FIG. 8. This analysis showed that seven ofthese peptides significantly increased the expression of HLA A2 on T2cells TABLE 3 Identification of Potential HLA A2 Binding Peptides withingp85 and gp350* Score (Estimate half-time disassociation from HLARanking Residue Peptide Sequence A2 allele) gp85 peptides 1 177-185FLMGTYKRV (SEQ ID NO:12) 1775.663 2 317-325 WLAKSFFEL (SEQ ID NO:13)1082.903 3 672-680 GLYEERAHV (SEQ ID NO:14) 912.522 4 685-693 ILYFIAFAL(SEQ ID NO:15) 674.026 5  2-10 QLLCVFCLV (SEQ ID NO:16) 488.951 6225-233 SLVIVTTFV (SEQ ID NO:17) 382.536 7 681-689 VLAIILYFI (SEQ IDNO:18) 224.537 8 684-692 IILYFIAFA (SEQ ID NO:19) 196.407 9 542-550LMIIPLINV (SEQ ID NO:20) 181.738 10 1-9 MQLLCVFCL (SEQ ID NO:21) 181.73811  7-15 FCLVLLWEV (SEQ ID NO:22) 133.298 12 658-666 YLLLTTNGT (SEQ IDNO:23) 126.883 13 420-428 TLFIGSHVV (SEQ ID NO:24) 105.510 gp350peptides 1 871-879 VLTLLLLLV (SEQ ID NO:25) 271.948 2 152-160 LIPETVPYI(SEQ ID NO:26) 126.481 3 863-871 VLQWASLAV (SEQ ID NO:27) 118.238 4875-883 LLLLVMADC (SEQ ID NO:28) 71.872 5 67-75 QLTPHTKAV (SEQ ID NO:29)69.552 6 861-869 MLVLQWASL (SEQ ID NO:30) 61.737 7 873-881 TLLLLLVMA(SEQ ID NO:31) 42.278

[0131] suggesting that these peptides might be potential HLAA2-restricted epitopes.

[0132] Recognition of the gp85 and gp350 Peptide Epitopes by IMEffectors Ex Vivo

[0133] The seven HLA A2-binding peptides, which included four peptidesfrom gp85 and three peptides from gp350 were next tested for CTLrecogniton by effectors from IM patients. In addition, we also includedan HLA A2-restricted CTL epitope from EBV latent mambrane protein (LMP1)as a positive control (38). PBMCs from three HLA A2-positive IMpatients, SB, LP and MG were resuspended in IL2-supplemented growthmedium and used as effectors in a standard 51Cr-release assay againstHLA-matched PHA blasts sensitized with the gp85, gp350 or LMP1 peptides.Representative data from two different experiments is shown in FIG.9(A-C). Effectors from all three IM patients showed clear recognition ofthe reference LMP1 peptide (YLQQNWWTL (SEQ ID NO: 6)) consistent withour earlier finding that this peptide is recognized by EBV-specificCTLs. More importantly, these IM patients also showed strong recognitionof target cells sensitized with selected gp85 or gp350 peptides.Interestingly, each of these individuals showed a distinct pattern ofreactivity against these peptides. IM patient SB showed strongreactivity against peptides SLVIVTTFV (SEQ ID NO: 17) (gp85) andVLQWASLAV (SEQ ID NO: 27) (gp350) (FIG. 9A), while the LP and MGeffectors recognised target cells preloaded with peptides LMIIPLINV (SEQID NO: 20) (gp85) and VLQWASIAV (SEQ ID NO: 27) (gp350) (FIG. 9(B-C)).Furthermore, ex vivo effectors from patient LP also recognised targetcells infected with Vacc.gp350 and Vacc.gp85 (FIG. 9D).

[0134] In Vitro Expansion of gp85 and gp350 Peptide Epitope ReactiveCTLs

[0135] The data presented above clearly demonstrate that gp85 and gp350include CTL determinants which can bind HLA A2 molecules and areefficiently recognised by ex vivo effectors from IM patients. Todetermine whether gp85- or gp350-reactive CTLs can be detected followingrecovery from IM, PBMCs from the two donors SB and LP were collected at24-36 months post IM respectively and were stimulated with T2 cellspresensitized with each of the gp85 and gp350 peptides which showedstrong HLA A2 binding. On day 18. these CTL effector were tested againstpeptide-sensitized autologous PHA blasts. Representative data frompolyclonal CTLs from donor SB are presented in FIG. 10. CTL effectorsfrom donor SB not only showed strong reactivity against peptidesSLVIVTTFV (SEQ ID NO: 17) and VLQWASIAV (SEQ ID NO: 27) but alsorecognized two other peptides from gp85 (LMIIPLINV (SEQ ID NO: 20) andTLFIGSHVV (SEQ ID NO: 24)). Donor LP also showed a similar pattern ofCTL lysis. Thus peptide TLFIGSHVV (SEQ ID NO: 24) was a target forEBV-specific CTL recogntion in the memory response of these A2-positiveindividuals, but this response was not detectable with ex vivo effectorsduring acute infection. Another important point which needs to behighlighted here is that our attempts to activate gp85- orgp350-specific CTLs with autologus LCLs as stimulators wereunsuccessful. This result is not surprising since it is well establishedthat in latently infected B cells, gp350 or gp85 antigens are poorlyexpressed. The LCL-stimulated polyclonal T cell lines from these donorsstrongly reactive against latent antigens (data not shown). Thisobseravtion is consistent with our earlier studies which showed that CTLresponses in healthy virus carriers is often dominated by reactivity tolatent antigens (6). Another explanation for an inability to detectgp350- or gp85-specific CTL reactivity following stimulation with theautologous LCLs is that these responses may constitute a minor componentof the total virus specific CTL response in healthy virus carriers.Indeed, limiting dilution analysis for CTL precursors specific for thegp350 or gp85 peptide epitopes in post IM donors SB and LP showedprecursor frequencies of >1/50,000, while precursor frequencies for CTLsthat recognise CTL epitopes within the latent antigens were between1/4,000-1/15,000 (data not shown).

[0136] Immunization of HLA A2/Kb Mice with gp85 and gp350 PeptideEpitopes Induces Specific CTL Response

[0137] Having established that gp85 and gp350 includes CTL epitopes, weextended our studies to explore the possibility of using these peptideepitopes to induce specific CTL response in vivo. HLA A2/Kb transgenicmice were used as an experimental model to address this issue. Thesemice express a chimeric class I molecule composed of the alpha 1 & 2domains of the human A*0201 allele and the alpha 3 domain of the mouseH-2Kb class I molecules. These animals were immunized subcutaneouslywith gp350 or gp85 CTL epitopes emulsified in IFA together with TetanusToxoid as a source of help. The SLVIVTTFV (SEQ ID NO: 17) and TLFIGSHVV(SEQ ID NO: 24) peptides from gp85 and the VLQWASLAV (SEQ ID NO: 27)peptide from gp350 were used for immunisation. Two weeks followingimmunization, specific CTL response was assessed in each mouse usingsplenocytes or pooled inguinal lymph node cells as effectors. Datapresented in FIG. 11(A-C) demonstrate that peptide epitopes from gp85(SLVIVTTFV (SEQ ID NO: 17) and TLFIGSHVV (SEQ ID NO: 24)) and gp350(VLQWASLAV (SEQ ID NO: 27)) induced strong CTL response in splenocytes.Interestingly, CTLs activated from splenocytes with peptide TLFIGSHVV(SEQ ID NO: 24) consistently showed strong lysis of targets, whilesplenocytes from SLVIVTTFV (SEQ ID NO: 17) and VLQWASLAV (SEQ ID NO: 27)immunized mice showed variable in vitro CTL lysis. A strong specific CTLactivity was also noticed in pooled lymphocytes from inguinal lymphnodes (FIG. 11D).

[0138] Prior Immunisation of HLA A2/Kb Mice with gp85 or gp350 CTLEpitopes Affords Protection Against Recombinant Vaccinia Virus Challenge

[0139] Four weeks after peptide immunization with gp85 or gp350 CTLepitopes, HLA A2/K b mice were challenged with 10⁷ pfu of recombinantvaccinia virus encoding either gp85 or gp350. After four days ofchallenge, these animals were sacrificed and vaccinia titres measured inboth ovaries by plaque assay on confluent CV1 cells. Data from one suchexperiment is presented in FIG. 12. Animals immunised with gp85 andgp350 epitopes showed very low to undetectable virus in their ovaries,while in naive mice very high titres of vaccinia virus were detected.This protection correlated with strong induction of epitope-specific CTLresponses detected in the splenocytes and lymph node cells collectedfour weeks after primary peptide vacciniation in HLA A2/Kb transgenicmice.

Discussion

[0140] There is increasing interest in formulating an effective vaccineagainst EBV, designed to not only limit the outgrowth of latentlyinfected B cells in healthy individuals but to also block thedevelopment of many EBV-associated malignancies such as Burkitt'slymphoma (BL), nasopharyngeal carcinoma (NPC) and Hodgkin's disease(HD). In western societies, the principle aim of such a vaccine would beto protect from IM. In this context, virus load (a large dose of orallytransmitted virus and/or overexpansion of the virus-transformed B cellpool beyond a critical threshold) may be a critical determinant ofdisease risk (7). Therefore, a vaccine capable of either blockingprimary EBV infection or significantly reducing the EBV load duringprimary infection may be adequate to avert clinical symptoms. A similarvaccine will also be able to reduce the immediate risk oflymphoproliferative disease in transplant patients receivingimmunosuppressive therapy. On the other hand, EBV-associatedmalignancies such as BL, NPC, and HD arise in patients years after theirprimary infection, and protection from these longer-term consequenceswould require a vaccine that ideally confers sterile immunity andprevents the establishment of the carrier state.

[0141] EBV structural antigens, primarily gp350, have long beenconsidered as the potential candidates for an EBV vaccine. Thesuggestion that gp350 is a likely vaccine candidate was based initiallyupon the observation that this glycoprotein is the principal target ofthe virus-neutralizing antibody response (41). A number of recombinantformulations of gp350, either presented as a subunit antigen orexpressed from recombinant viral vectors, designed to induce high titreneutralizing antibodies, have shown significant protection againstEBV-induced B cell lymphomas in cotton-top tamarins (31). However,development of neutralizing antibodies in vaccinated animals does notalways shows limited correlation with protection from EBV infection,although recent results have suggested a role for gp350-specific CTLs inthis protection (34). If the latter suggestion is correct, it isimportant to identify the potential CTL determinants within EBVstructural proteins since it is now well established that immunizationwith whole viral proteins is unable to elicit an efficient CTL response.Moreover, a vaccine based on CTL epitopes provides an opportunity toinclude determinants not only from gp350 but also from other structuralantigens, such as gp85. To address this issue we have used a novelprotocol to successfully identify CTL epitopes within gp350 and gp85. Inthe first set of experiments we identified HLA A2 binding peptideswithin gp350 and gp85. Subsequent experiments were focussed on IMpatients with the HLA A2 allele. Using ex vivo primary effectors, weobserved strong reactivity to three different gp350 and gp85 peptides.Interestingly, individual IM patients showed distinct patterns ofreactivity to each of these peptides. Strong reactivity against peptidesSLVIVTTFV (SEQ ID NO: 17) (gp85) and VLQWASLAV (SEQ ID NO: 27) (gp350)was observed with ex vivo effectors from patient SB, while the LP and MGeffectors recognized target cells preloaded with LMIIPLINV (SEQ ID NO:20) (gp85) and VLQWASLAV (SEQ ID NO: 27) (gp350) peptides. Moreimportantly, ex vivo effectors from patient LP also recognised targetcells infected with Vacc.gp350 and Vacc.gp85. Interestingly, the levelof ex vivo CTL lysis directed to epitopes from structural antigens wasconsistently higher than those seen in the same assays against HLAA2-restricted CTL epitopes from a latent antigen. These results areconsistent with recent observations by Steven and colleagues (42) thatex vivo CTL reactivity to lytic antigens in IM patients is significantlyhigher compared to latent antigens.

[0142] In the next set of experiments, we explored the possibility ofdetecting structural antigen-specific CTL responses in individualsfollowing resolution of IM symptoms. This follow up analysis was carriedout 24-36 months post acute IM. Our initial attempts to isolate gp350-or gp85-specific CTLs from post IM donors by stimulating with theautologous LCL were unsuccessful. Subsequently we used peptide loaded T2cells as stimulators to generate gp350- and gp85-specific CTLs. We haverecently shown that this method can be successfully used to raise lowfrequency EBV-specific CTL precursors (38). Stimulation of PBMC fromdonors SB and LP raised strong CTL responses to the gp85 and gp350 CTLepitopes. Both donors SB and LP not only showed reactivity againstpeptides SLVIVTTFV (SEQ ID NO: 17), LMIIPLINV (SEQ ID NO: 20) andVLQWASLAV (SEQ ID NO: 27) but also recognized another peptide from gp85,TLFIGSHVV (SEQ ID NO: 24). It is interesting to note here that bothdonors showed no ex vivo CTL reactivity to TLFIGSHVV (SEQ ID NO: 24)during acute IM. One of the important conclusions drawn from theseanalyses is that, following recovery from acute IM, there is asignificant reduction in CTL precursors to the structural antigens, andthe response becomes dominated by CTL reactive to the latent antigens.Indeed, limiting dilution analysis for CTL precursors specific for thegp350 or gp85 peptide epitopes in donors SB and LP post IM showedfrequencies of >1/50,000, while precursor frequencies for CTL epitopeswithin latent antigens were between 1/4,000-1/15,000.

[0143] The detection of a strong ex vivo CTL response in IM patients tothe structural antigens has important implications for any futurevaccine design. As mentioned above, to date, the major emphasis ofvaccine design based on EBV structural antigens has been directedtowards generating a strong neutralizing antibody response. However,these neutralising antibody response fail to correlate with protectionagainst EBV-induced polyclonal lymphomas in cotton-top marmosets.Nevertheless, it is possible that this protection is mediated bystructural antigen-specific CTL responses. To address this issue, weemployed an experimental animal model system to determine whether gp350or gp85 CTL epitope immunized transgenic mice, expressing the human HLAA2 antigen, are capable of (a) generating structural antigen-specificCTL responses and (b) reducing infection with a recombinant vacciniavirus infection expressing the gp350 or gp85 antigen. These mice notonly showed induction of a strong CTL response following immunizationbut also acquired strong resistance to virus infection. It is importantto mention here that although this experiment does not allow any firmconclusions on the efficacy of a gp350 and/or gp85 CTL epitope basedvaccine in humans, it does clearly show that CTL epitopes from the EBVstructural antigens can be used as immunogens to induce an efficient CTLresponse in vivo. Moreover, this approach also overcomes limitations ofwhole gp350 or gp85 proteins which might be inefficient at eliciting CTLresponses in humans. Obviously one of the possible obstacles of anyepitope-based approach to vaccination in humans is HLA polymorphismbecause epitope choice is allele-specific. However, this obstacle mightbe overcome using appropriate mixtures of synthetic peptide epitope orby constructing vectors to express polypeptides in which the relevantepitope sequences are linearly joined together. Indeed, earlier studiesfrom our laboratory have shown that if such an EBV polyepitope sequenceis expressed within cells from a recombinant vaccinia vector, all of theconstituent epitopes are efficiently presented for CTL recognition (43),indicating the potential of this approach as a vaccine strategy. Morerecently, work in a murine model has also shown that each of several CTLepitopes combined in a polyepitope construct was capable of eliciting aCTL response in vivo and could protect the animals from subsequentchallenge (44). In the long term, it may be possible to combine CTLepitopes from the EBV structural antigens with latent antigen epitopesgenerating a chimeric protein that fuses the important immunogenicdeterminants from the two different types of antigens to design aneffective vaccine.

[0144] It will be appreciated by persons skilled in the art thatnumerous variations and/or modifications may be made to the invention asshown in the specific embodiments without departing from the spirit orscope of the invention as broadly described. The present embodimentsare, therefore, to be considered in all respects as illustrative and notrestrictive.

1 33 1 9 PRT Epstein Barr Virus 1 Tyr Leu Leu Glu Met Leu Trp Arg Leu 15 2 9 PRT Epstein Barr Virus 2 Leu Leu Leu Ala Leu Leu Phe Trp Leu 1 5 39 PRT Epstein Barr Virus 3 Leu Leu Val Asp Leu Leu Trp Leu Leu 1 5 4 9PRT Epstein Barr Virus 4 Leu Leu Leu Ile Ala Leu Trp Asn Leu 1 5 5 9 PRTEpstein Barr Virus 5 Trp Leu Leu Leu Phe Leu Ala Ile Leu 1 5 6 9 PRTEpstein Barr Virus 6 Tyr Leu Gln Gln Asn Trp Trp Thr Leu 1 5 7 9 PRTEpstein Barr Virus 7 Thr Leu Leu Val Asp Leu Leu Trp Leu 1 5 8 9 PRTEpstein Barr Virus 8 Leu Leu Trp Leu Leu Leu Phe Leu Ala 1 5 9 9 PRTEpstein Barr Virus 9 Ile Leu Leu Ile Ile Ala Leu Tyr Leu 1 5 10 9 PRTEpstein Barr Virus 10 Val Leu Phe Ile Phe Gly Cys Leu Leu 1 5 11 9 PRTEpstein Barr Virus 11 Arg Leu Gly Ala Thr Ile Trp Gln Leu 1 5 12 9 PRTEpstein Barr Virus 12 Phe Leu Met Gly Thr Tyr Lys Arg Val 1 5 13 9 PRTEpstein Barr Virus 13 Trp Leu Ala Lys Ser Phe Phe Glu Leu 1 5 14 9 PRTEpstein Barr Virus 14 Gly Leu Tyr Glu Glu Arg Ala His Val 1 5 15 9 PRTEpstein Barr Virus 15 Ile Leu Tyr Phe Ile Ala Phe Ala Leu 1 5 16 9 PRTEpstein Barr Virus 16 Gln Leu Leu Cys Val Phe Cys Leu Val 1 5 17 9 PRTEpstein Barr Virus 17 Ser Leu Val Ile Val Thr Thr Phe Val 1 5 18 9 PRTEpstein Barr Virus 18 Val Leu Ala Ile Ile Leu Tyr Phe Ile 1 5 19 9 PRTEpstein Barr Virus 19 Ile Ile Leu Tyr Phe Ile Ala Phe Ala 1 5 20 9 PRTEpstein Barr Virus 20 Leu Met Ile Ile Pro Leu Ile Asn Val 1 5 21 9 PRTEpstein Barr Virus 21 Met Gln Leu Leu Cys Val Phe Cys Leu 1 5 22 9 PRTEpstein Barr Virus 22 Phe Cys Leu Val Leu Leu Trp Glu Val 1 5 23 9 PRTEpstein Barr Virus 23 Tyr Leu Leu Leu Thr Thr Asn Gly Thr 1 5 24 9 PRTEpstein Barr Virus 24 Thr Leu Phe Ile Gly Ser His Val Val 1 5 25 9 PRTEpstein Barr Virus 25 Val Leu Thr Leu Leu Leu Leu Leu Val 1 5 26 9 PRTEpstein Barr Virus 26 Leu Ile Pro Glu Thr Val Pro Tyr Ile 1 5 27 9 PRTEpstein Barr Virus 27 Val Leu Gln Trp Ala Ser Leu Ala Val 1 5 28 9 PRTEpstein Barr Virus 28 Leu Leu Leu Leu Val Met Ala Asp Cys 1 5 29 9 PRTEpstein Barr Virus 29 Gln Leu Thr Pro His Thr Lys Ala Val 1 5 30 9 PRTEpstein Barr Virus 30 Met Leu Val Leu Gln Trp Ala Ser Leu 1 5 31 9 PRTEpstein Barr Virus 31 Thr Leu Leu Leu Leu Leu Val Met Ala 1 5 32 9 PRTEpstein Barr Virus 32 Tyr Phe Leu Glu Ile Leu Trp Gly Leu 1 5 33 9 PRTEpstein Barr Virus 33 Tyr Leu Leu Glu Ile Leu Trp Arg Leu 1 5

1. A cytotoxic Epstein-Barr virus (EBV) T-cell epitope the epitope beingderived from an EBV structural antigen.
 2. A cytotoxic EBV T-cellepitope as claimed in claim 1 wherein the EBV structural antigen is gp85or gp350.
 3. A cytotoxic T-cell epitope, the epitope being selected fromthe group consisting of YLLEMLWRL (SEQ ID NO: 1), YFLEILWGL (SEQ ID NO:32), YLLEILWRL (SEQ ID NO: 33), YLQQNWWTL (SEQ ID NO: 6), LLLALLFWL (SEQID NO: 2), LLVDLLWLL (SEQ ID NO: 3), LLLIALWNL (SEQ ID NO: 4), WLLLFLAIL(SEQ ID NO: 5), TLLVDLLWL (SEQ ID NO: 7), LLWLLLFLA (SEQ ID NO: 8),ILLIIALYL (SEQ ID NO: 9), VLFIFGCLL (SEQ ID NO: 10), RLGATIWQL (SEQ IDNO: 11), ILYFIAFAL (SEQ ID NO: 15), SLVIVTTFV (SEQ ID NO: 17), LMIIPLINV(SEQ ID NO: 20), TLFIGSHVV (SEQ ID NO: 24), LIPETVPYI (SEQ ID NO: 26),VLQWASLAV (SEQ ID NO: 27) and QLTPHTKAV (SEQ ID NO: 29).
 4. A subunitvaccine including a cytotoxic Epstein-Barr virus (EBV) T-cell epitope asclaimed in claim 1 or claim
 2. 5. A subunit vaccine including at leastone T-cell epitope selected from the group consisting of YLLEMLWRL (SEQID NO: 1), YFLEILWGL (SEQ ID NO: 32), YLLEILWRL (SEQ ID NO: 33),YLQQNWWTL (SEQ ID NO: 6), LLLALLFWL (SEQ ID NO: 2), LLVDLLWLL (SEQ IDNO: 3), LLLIALWNL (SEQ ID NO: 4), WLLLFLAIL (SEQ ID NO: 5), TLLVDLLWL(SEQ ID NO: 7), LLWLLLFLA (SEQ ID NO: 8), ILLIIALYL (SEQ ID NO: 9),VLFIFGCLL (SEQ ID NO: 10), RLGATIWQL (SEQ ID NO: 11), ILYFIAFAL (SEQ IDNO: 15), SLVIVTTFV (SEQ ID NO: 17), LMIIPLINV (SEQ ID NO: 20), TLFIGSHVV(SEQ ID NO: 24), LIPETVPYI (SEQ ID NO: 26), VLQWASLAV (SEQ ID NO: 27)and QLTPHTKAV (SEQ ID NO: 29).
 6. A subunit vaccine as claimed in claim5 wherein the epitope is selected from the group consisting of YLLEMLWRL(SEQ ID NO: 1), YLQQNWWTL (SEQ ID NO: 6), YFLEILWGL (SEQ ID NO: 32),YLLEILWRL (SEQ ID NO: 33), SLVIVTTFV (SEQ ID NO: 17), LMIIPLINV (SEQ IDNO: 20), TLFIGSHVV (SEQ ID NO: 24), and VLQWASLAV(SEQ ID NO: 27).
 7. Asubunit vaccine as claimed in any one of claims 4 to 6 wherein thevaccine further includes at least one antigen to which the individualwill mount an anamnestic response in addition to the at least onecytotoxic T-cell epitope.
 8. A subunit vaccine as claimed in claim 7wherein the at least one antigen is selected from the group consistingof tetanus toxoid, diphtheria toxoid, Bordetella pertussis antigens,poliovirus antigens, purified protein derivative (PPD), gp350 protein,helper epitopes and combinations thereof.
 9. A subunit vaccine asclaimed in claim 8 wherein the at least one antigen is tetanus toxoid.10. A subunit vaccine as claimed in any one of claims 4 to 9 in whichthe vaccine includes a water-in-oil formulation.
 11. An isolated nucleicacid sequence encoding a cytotoxic Epstein-Barr virus (EBV) T-cellepitope as claimed in claim 1 or claim
 2. 12. An isolated nucleic acidsequence encoding at least one of the cytotoxic T-cell epitopes selectedfrom the group consisting of YLLEMLWRL (SEQ ID NO: 1), YFLEILWGL (SEQ IDNO: 32), YLLEILWRL (SEQ ID NO: 33), YLQQNWWTL (SEQ ID NO: 6), LLLALLFWL(SEQ ID NO: 2), LLVDLLWLL (SEQ ID NO: 3), LLLIALWNL (SEQ ID NO: 4),WLLLFLAIL (SEQ ID NO: 5), TLLVDLLWL (SEQ ID NO: 7), LLWLLLFLA (SEQ IDNO: 8), ILLIIALYL (SEQ ID NO: 9), VLFIFGCLL (SEQ ID NO: 10), RLGATIWQL(SEQ ID NO: 11), ILYFIAFAL (SEQ ID NO: 15), SLVIVTTFV (SEQ ID NO: 17).LMIIPLINV (SEQ ID NO: 20), TLFIGSHVV (SEQ ID NO: 24), LIPETVPYI (SEQ IDNO: 26), VLQWASLAV (SEQ ID NO: 27) and QLTPHTKAV (SEQ ID NO: 29).
 13. Anisolated nucleic acid sequence as claimed in claim 12 wherein theepitope is selected from the group consisting of YLLEMLWRL (SEQ ID NO:1), YLQQNWWTL (SEQ ID NO: 6), YFLEILWGL (SEQ ID NO: 32), YLLEILWRL (SEQID NO: 33), SLVIVTTFV (SEQ ID NO: 17), LMIIPLINV (SEQ ID NO: 20),TLFIGSHVV (SEQ ID NO: 24), and VLQWASLAV (SEQ ID NO: 27).
 14. A vectorincluding a nucleic acid sequence as claimed in any one of claims 11 to13.
 15. A vector as claimed in claim 14 in which the vector is abacteria, preferably Salmonella spp.
 16. A vector as claimed in claim 14in which the vector is a virus. preferably Adenovirus. Retrovirus orVaccinia, and most preferably Modified Vaccinia Ankara.
 17. An isolatedpolypeptide, the polypeptide including at least one EBV CTL epitope asclaimed in any one of claims 1 to
 3. 18. A method of preparing acomposition for use in inducing CTLs in a subject, the method includingadmixing at least one epitope as claimed in any one of claims 1 to 3with at least one pharmaceutically acceptable carrier, diluent orexcipient.
 19. A method of reducing the risk of EBV infection in asubject which method includes administering to the subject an effectiveamount of: (1) at least one CTL epitope as claimed in any one of claims1 to 3; (2) a subunit vaccine as claimed in any one of claims 4 to 10;(3) a nucleic acid sequence as claimed in any one of claims 11 to 13;(4) a vector as claimed in any one of claims 14 to 16; or (5) apolypeptide as claimed in claim
 17. 20. A method of treating orpreventing nasopharyngeal carcinoma or Hodgkin's disease in a subjectwhich method includes administering to the subject an effective amountof at least one CTL epitope derived from an EBV structural or latentantigen.
 21. A method according to claim 20 wherein the EBV structuralantigen is gp85 or gp350.
 22. A method according to claim 20 wherein theEBV latent antigen is LMP1 or LMP2.
 23. A method according to claim 20wherein at least one CTL epitope selected from the group consisting ofYLLEMLWRL (SEQ ID NO: 1), YFLEILWGL (SEQ ID NO: 32), YLLEILWRL (SEQ IDNO: 33), YLQQNWWTL (SEQ ID NO: 6), LLLALLFWL (SEQ ID NO: 2),LLVDLLWLL(SEQ ID NO: 3), LLLIALWNL (SEQ ID NO: 4), WLLLFLAIL (SEQ ID NO:5), TLLVDLLWL (SEQ ID NO. 7). LLWLLLFLA (SEQ ID NO: 8), ILLIIALYL (SEQID NO: 9), VLFIFGCLL (SEQ ID NO: 10), RLGATIWQL (SEQ ID NO: 11),ILYFIAFAL (SEQ ID NO: 15), SLVIVTTFV (SEQ ID NO: 17), LMIIPLINV (SEQ IDNO: 20), TLFIGSHVV (SEQ ID NO: 24), LIPETVPYI (SEQ ID NO: 26), VLQWASIAV(SEQ ID NO: 27) and QLTPHTKAV (SEQ ID NO: 29), is administered to thesubject.
 24. A method of treating or preventing growth of NPC cells in asubject in need thereof which method includes administering to thesubject at least one CTL epitope derived from an EBV structural orlatent antigen.
 25. A method according to claim 24 wherein the EBVstructural antigen is gp85 or gp350.
 26. A method according to claim 24wherein the EBV latent antigen is LMP1 or LMP2.
 27. A method accordingto claim 27 wherein at least one CTL epitope selected from the groupconsisting of YLLEMLWRL (SEQ ID NO: 1), YFLEILWGL (SEQ ID NO: 32),YLLEILWRL (SEQ ID NO: 33), YLQQNWWTL (SEQ ID NO: 6), LLLALLFWL (SEQ IDNO: 2), LLVDLLWLL (SEQ ID NO: 3), LLLIALWNL (SEQ ID NO: 4), WLLLFLAIL(SEQ ID NO: 5), TLLVDLLWL (SEQ ID NO: 7), LLWLLLFLA (SEQ ID NO: 8),ILLIIALYL (SEQ ID NO: 9), VLFIFGCLL (SEQ ID NO: 10), RLGATIWQL (SEQ IDNO: 11), ILYFIAFAL (SEQ ID NO: 15), SLVIVTTFV (SEQ ID NO: 17), LMIIPLINV(SEQ ID NO: 20), TLFIGSHVV (SEQ ID NO: 24), LIPETVPYI (SEQ ID NO: 26),VLQWASLAV (SEQ ID NO: 27) and QLTPHTKAV (SEQ ID NO: 29), is administeredto the subject.
 28. A method of treating or preventing the growth of NPCor HD cells in a first subject which method includes transferring to thefirst subject EBV-specific CTLs which recognise NPC or HD cells.
 29. Amethod as claimed in claim 28 wherein the EBV-specific CTLs are obtainedfrom the first subject by in vitro stimulation of CTLs by exposure toEBV CTL epitopes.
 30. A method as claimed in claim 28 wherein theEBV-specific CTLs are obtained from a second subject, wherein the secondsubject is infected with EBV but does not have NPC or HD.
 31. A methodas claimed in claim 26 wherein the EBV-specific CTLs are LMP1 and/orLMP2-specific CTLs.
 32. A method of reducing the risk of infectiousmononucleosis or post transplantation lymphoproliferative disease in asubject which method includes administering to the subject an effectiveamount of: (1) at least one CTL epitope as claimed in any one of claims1 to 3; (2) a subunit vaccine as claimed in any one of claims 4 to 10;(3) a nucleic acid sequence as claimed in any one of claims 11 to 13;(4) a vector as claimed in any one of claims 14 to 16; or (5) apolypeptide as claimed in claim 17.